Project Summary Diabetes mellitus (DM) is a chronic metabolic disorder leading to progressive secondary complications such as blindness, heart failure, kidney failure and stroke. Despite the success of pharmacological management of DM, diabetic retinopathy (DR) remains a leading cause of blindness worldwide. Clinical trials have demonstrated that treatment of dyslipidemia, in addition to hyperglycemia, plays an important role in slowing DR progression. However, blood lipid profiles are poorly correlated with clinical outcome. These findings suggest that cell and/or tissue specific lipid changes are important in disease progression. The retina is a highly metabolically active tissue whose metabolic demands are matched by a rich vascular supply, high oxygen tension and dense mitochondria. Mitochondrial dysfunction plays a central role in DR progression. Changes to mitochondrial structure and function have been shown to precede histological changes and increased mitochondrial metabolic flux has been proposed as a ?unifying mechanism? to explain cellular damage secondary to hyperglycemia. In addition to glucotoxicity, lipotoxicity has been shown to potentiate reactive oxygen species (ROS) generation in retinal endothelial cells, emphasizing the importance of dyslipidemia in DR progression. Histological hallmarks of microvasculature changes in DR include loss of pericytes, blood-retinal barrier breakdown, and endothelial cell apoptosis leading to formation of acellular capillaries. The Busik group has previously demonstrated the protective effect of ceramide depletion, via acid sphingomyelinase (ASM) knock out, on early histological changes in mouse models of DR. ASM is an enzyme of the ceramide salvage pathway, which hydrolyzes sphingomyelin to ceramide and phosphocholine. Ceramides and other sphingolipid species, along with their synthetic enzymes, have been localized to mitochondria and shown to cause ROS generation, mitochondrial outer membrane permeability and electron transport chain inhibition. Preliminary data shows the accumulation of mitochondrial ceramide in diabetic rodent retina which is abrogated in ASM knock out mice. In vitro studies have shown the ability of short chain ceramides to form supramolecular structures capable of transporting proteins through planar lipid bilayers and work on isolated mitochondria has implicated these sphingolipid species in playing a key role in MOMP leading to cytochrome c release and apoptosis. The mechanistic details underlying ceramide-induced microvascular dysfunction in DR remain to be determined. The objective of this proposal is to define the role of diabetes-induced changes in mitochondrial sphingolipid metabolism in retinal endothelial cell damage in DR.